Date of Award
Doctor of Philosophy (PhD)
The biosynthetic mechanisms responsible for the segregation of secreted proteins and for the insertion of integral membrane proteins have been examined in light of the signal hypothesis. Model systems for the study of the biosynthesis of these different classes of proteins, as well as for the study of specific events common to both, have been developed. Their common as well as distinctive features have been probed by a variety of different experimental approaches involving cell free protein synthesizing systems supplemented with heterologous microsomal membranes either during or after translation: a) proteolytic enzymes, SDS-PAGE and autoradiography have been used to characterize various biosynthetic forms of bovine pituitary growth hormone and prolactin, vesicular stomatitis virus glycoprotein, rat mammary α-lactalbumin and chicken oviduct ovalbumin. b) amino terminal sequence analysis has been performed on various forms of these proteins synthesized in cell free systems. The sequence data presented demonstrate that nascent precursors to both secretory and membrane proteins contain "signal peptides" at the NH2 terminus; which display common structural features. In most--but not all--cases of both classes of proteins the signal sequence is proteolytically cleaved (correctly) during the process of insertion into microsomal membranes prior to chain completion. The transmembrane orientation of the inserted cleaved and glycosylated membrane protein is identical to that of the authentic glycoprotein from virions, while the secreted proteins are completely segregated within the vesicular spaces of the heterologous microsomal membranes. c) Nascent chain competition experiments were performed in order to functionally characterize and contrast these various mechanisms. The data suggest that an initially common pathway is involved in the biogenesis of secretory proteins (both with and without cleaved signals) and at least one class of integral membrane proteins.
Lingappa, Vishwanath R., "Early Events in the Biosynthesis and Subcellular Consignment of Secretory and Membrane Proteins" (1979). Student Theses and Dissertations. 488.